Liquid Matrices for Analyses by UV-MALDI Mass Spectrometry

Matrix-assisted laser desorption ionization (MALDI) (1,2) is one of the most used ionization techniques in mass spectrometry (MS) for the analysis of large biomolecules and has been extremely successful in fields such as proteomics (3–5). In MALDI-MS, a matrix, typically a weak organic acid such as α-cyano-4-hydroxycinnamic acid (CHCA) (6) or 2,5-dihydroxybenzoic acid (DHB) (7), is used to enable the production of stable gas-phase ions of an analyte of interest, enabling its MS analysis. Most commonly, the matrix material is dissolved in a suitable solvent and mixed with the analyte either on or off the target. Loss of the solvent on the sample target through evaporation during the sample preparation step ideally results in matrix–analyte cocrystallization. As a consequence of the subsequent MALDI process that is induced by short, focused laser pulse irradiation (typically <10 ns and <108 W/cm2 ), ablation of the solid MALDI sample leads to desorption and ionization of both matrix and analyte ions. The matrix absorbs the energy without imparting excessive internal energy to the analyte, thereby limiting fragmentation. The desorption–ionization process is essentially a thermodynamic and physicochemical process, and is best described as a fast solid-state to gas-phase transition triggered by the absorption of the laser energy by the matrix (Figure 1). This is followed by a jet expansion of the resulting gas plume into the vacuum (8–10). The exact mechanism that causes the creation of the gas-phase analyte ions is not yet fully understood, and a variety of possibilities have been proposed (8–10). The most probable route for ionization of biomolecules, such as polypeptides, is that of energy pooling through singlet–singlet annihilation. This produces free radical matrix ions (11) followed in the positive ion mode by proton transfer (8,10) between the positive matrix radical ions or resulting protonated matrix ions and the analyte molecules, thereby generating the protonated analyte ions.

As expected, the matrix used in MALDI-MS has a considerable effect on the peak intensities, the relative abundance of biomolecules seen, the level of interfering ions observed, and fragmentation. Considerable work has gone into trying to define what makes a suitable matrix for MALDI. In general, however, the only steadfast rule is that the matrix molecules must absorb at the wavelength of light used and therefore absorb the energy without imparting excessive internal energy to the analyte. Other desirable qualities are vacuum stability, inertness, and the ability to generate a homologous matrix–analyte mix. In practice, most matrices cannot meet all of these requirements, so a compromise must be made based upon the application.

Compared with solid matrices, liquid matrices can have many advantages. The main advantage that a truly liquid matrix has over a solid matrix is that of homogenous sample mixing and a simple and even morphology. With a solid crystalline matrix, the analyte might not be distributed evenly throughout the crystals, the laser bores into the crystal on successive shots, or the laser desorption position needs to be changed frequently — all potentially leading to significant changes in ion signal abundances and inefficient sample consumption. Searching for "hot spots" is often required, and as each crystal is reduced in size, the distance the analyte travels increases, affecting mass resolution and accuracy. With a liquid matrix, a near-uniform surface is presented; because the liquid is self-healing and renewing, the peak abundance remains relatively stable once an equilibrium of renewal is achieved (12). This means that liquid matrices provide considerably better shot-to-shot reproducibility, as is required for quantitation (12).